Immunogenic peptides have been developed that elicit B and T cell responses to various strains of human immunodeficiency virus (HIV) (Palker et al, J. Immunol. 142:3612-3619 (1989), Haynes et al, Trans. Am. Assoc. Physician 106:31-41 (1993), Haynes et al, J. Immunol. 151:1646-1653 (1993), Haynes et al, AID Res. Human Retroviruses 11:211-221 (1995)) (see also WO 97/14436). These peptides consist of two components, each derived from noncontiguous regions of the HIV gp120 envelope protein. One envelope component consists of 16 amino acid residues from the fourth constant (C4) domain of HIV gp120, and includes a T-helper epitope (Cease et al, Proc. Natl. Acad. Sci. USA 84:4249-4253 (1987)). Linked to the carboxyl terminus of this gp120 C4 region peptide is a 23 amino acid segment from the third variable (V3) domain of gp120, that includes a B cell neutralizing antibody epitope for cell line-adapted HIV strains (Palker et al, J. Inmunol. 142:3612-3619 (1989), (Palker et al, Proc. Natl. Acad. Sci. USA 85:1932-1936 (1988), Rusche et al, Proc. Natl. Acad. Sci. USA 85:3198-3202)), a T-helper epitope (Palker et al, J. Immunol. 142:3612-3619 (1989)), and two cytotoxic T lymphopoietic (CTL) epitopes (Clerici et al, J. Immunol. 146:2214-2219 (1991), Safrit et al, 6th NCVDG Meeting, October 30 to Nov. 4, 1993)). In mice and rhesus monkeys, these C4-V3 hybrid peptides have induced antibodies that bind to native gp120 and neutralize the particular cell line-adapted strain of HIV from which the V3 segment was derived, as well as induce T helper cell proliferative responses and MHC Class I-restricted CTL responses that kill HIV or HIV protein expressing target cells (Palker et al, J. Immunol. 142:3612-3619(1989), Haynes et al, AID Res. Human Retroviruses 11:211-221 (1995)). Recently, it was shown that this gp120 peptide design can induce antibodies that neutralize primary HIV isolates and simian-human immunodeficiency viruses (SHIV) expressing primary HIV isolate envelopes (Liao et al, J. Virol. 74:254-263 (2000)). Moreover, in a challenge trial of this immunogen in rhesus monkeys, it was shown that C4-V3 peptides from the gp120 of the pathogenic SHIV 89.6P, induced neutralizing antibodies that prevented the fall in CD4 counts after challenge with SHIV 89.6P (Letvin et al, J. Virol. 75:4165-4175 (2001)). Therefore, anti-V3 antibodies can protect primates against primary isolate SHIV-induced disease.
A prototype polyvalent HIV experimental immunogen comprised of the conserved C4 region of gp120 and the V3 regions of HIV isolates MN, CANO(A), EV91 and RF has been constructed and has been found to be highly immunogenic in human clinical trials (Bartlett et al, AIDS 12:1291-1300 (1998), Graham et al, Abstract, AIDS Vaccine (2001)). Thus, understanding secondary and higher order structures of the components of this polyvalent immunogen, as well as defining strategies to optimize gp120 immunogen antigenicity, is important to HIV vaccine design efforts. In addition, recent data suggest that the HIV V3 region may be involved in regulating gp120 interactions with HIV co-receptors, CXC chemokine receptor 4 (CXCR4) and 13 chemokine receptor type 5 (CCR5) (Berger, AIDS Suppl. A:53-56 (1997)).
In previous studies, nuclear magnetic resonance (NMR) has been used to characterize conformations of the multivalent immunogen C4-V3 peptides in solution (de Lorimier et al, Biochemistry 33:2055-2062 (1994), Vu et al, Biochemistry 35:5158-5165 (1996), Vu et al, J. Virol. 73:746-750 (1999)). It as been found that the V3 segments of each of the four C4-V3 peptides displayed evidence of preferred solution conformations, with some features shared, and other features differing among the four peptides. The C4 segment, which is of identical sequence in all the peptides, showed in each case a tendency to adopt nascent helical conformations (de Lorimier et al, Biochemistry 33:2055-2062 (1994), Vu et al, Biochemistry 35:5158-5165 (1996), Vu et al, J. Virol. 73:746-750 (1999)).
The C4 domain of gp120 is part of the CD4 binding site, and antibodies recognizing the C4 sequence, block the binding of CD4 of gp120. However the C4 sequence as a peptide does not elicit antibodies that bind native gp120 (Palker et al, J. Immunol. 142:3612-3619 (1989), Haynes et al, J. Immunol. 151:1646-1653 (1993), Ho et al, J. Virol. 61:2024-2028 (1987), Robey et al, J. Biol. Chem. 270:23918-23921 (1995)). This led to the speculation that the nascent helical conformations exhibited by the C4 segment might reflect a conformation not native to HIV gp120. Amino-acid sequence homology between the gp120 C4 region and a human IgA CH1 domain has been noted (Maddon et al, Cell 47:333-348 (1986)). By comparison to the structure of mouse IgA (Segal et al, Proc. Natl. Acad. Sci. USA 71:4298-4302 (1974)), the C4-homologous region of IgA has a β strand secondary structure (de Lorimier et al, Biochemistry 33:2055-2062 (1994)). Therefore, while the C4 gp120 peptide in solution adopts nascent helical conformations, the native structure of this gp120 C4 region may be quite different (ie, in the context of gp 120 have a β strand secondary structure).
The present invention results, at least in part, from the results of a study with a three-fold purpose. First, C4-V3HIVRF peptides with amino acid substitutions designed to minimize C4 α-helical peptide conformation and promote β strand C4 secondary structures were constructed in order to induce anti-native gp120 antibodies with the modified C4 peptide. Second, tests were made to determine if any of these mutated C4-V3RF peptides would enhance gp120 V3 region peptide immunogenicity, and therefore augment anti-HI VRF gp120 V3 loop antibody responses. Finally, the solution conformers of each peptide studied immunologically were also solved using NMR to correlate peptide conformers with peptide immunogenicity.